Virulence Attributes and Host Response Assays for Determining Pathogenic Potential of Pseudomonas Strains Used in Biotechnology.

Tayabali AF, Coleman G, Nguyen KC - PLoS ONE (2015)

Bottom Line:
However, Pf and Pp strains were the most antibiotic resistant, with ciprofloxacin and colistin being the most effective of those tested.Serum amyloid A was elevated at ≥ 48 h post-exposure by only some Pa strains.No relationship was observed between strains and levels of peripheral leukocytes.

ABSTRACTPseudomonas species are opportunistically pathogenic to humans, yet closely related species are used in biotechnology applications. In order to screen for the pathogenic potential of strains considered for biotechnology applications, several Pseudomonas strains (P.aeruginosa (Pa), P.fluorescens (Pf), P.putida (Pp), P.stutzeri (Ps)) were compared using functional virulence and toxicity assays. Most Pa strains and Ps grew at temperatures between 28°C and 42°C. However, Pf and Pp strains were the most antibiotic resistant, with ciprofloxacin and colistin being the most effective of those tested. No strain was haemolytic on sheep blood agar. Almost all Pa, but not other test strains, produced a pyocyanin-like chromophore, and caused cytotoxicity towards cultured human HT29 cells. Murine endotracheal exposures indicated that the laboratory reference strain, PAO1, was most persistent in the lungs. Only Pa strains induced pro-inflammatory and inflammatory responses, as measured by elevated cytokines and pulmonary Gr-1 -positive cells. Serum amyloid A was elevated at ≥ 48 h post-exposure by only some Pa strains. No relationship was observed between strains and levels of peripheral leukocytes. The species designation or isolation source may not accurately reflect pathogenic potential, since the clinical strain Pa10752 was relatively nonvirulent, but the industrial strain Pa31480 showed comparable virulence to PAO1. Functional assays involving microbial growth, cytotoxicity and murine immunological responses may be most useful for identifying problematic Pseudomonas strains being considered for biotechnology applications.

pone.0143604.g004: Pulmonary Granulocyte Infiltration during Pseudomonas Exposure.Balb/c mice were endotracheally instilled with saline or 106 cfu of each Pseudomonas strain. At various times following exposure, animals were euthanized and lungs were harvested. Lungs were sectioned and stained with fluorescently tagged Ly6G antibody (inset shows example micrographs). Numbers of positively stained cells (red) were enumerated from nine micrograph fields (three fields from three mice for each treatment) and expressed as fold-change compared to those from saline-exposed mice. Asterisks indicate statistically different values compared to saline exposures, as determined using ANOVA and Dunnett’s Multiple Comparison Test (p < 0.05). Bacteria with significant differences of at least 2-fold are indicated with red boxes in the graph legends.

Mentions:
Certain Pseudomonas strains clearly induced a strong pro-inflammatory response, so pulmonary inflammation was monitored following exposure. This was done by enumerating granulocytes in lung tissue from micrographs such as those shown in insets of Fig 4. All Pa strains, but no other species were able to elevate numbers of pulmonary granulocytes between two and 48 h, and levels stabilized to control values by one week (Fig 4). The response appeared to be separated into two phases; an initial activation that occurred between two and 24 h (PAO1, Pa10752, Pa700370), and a delayed response at 48 h post-exposure (Pa31480, Pa700370, Pa700371). Furthermore, there was statistically significant drop in granulocyte levels at two hours for Pa10752 (2-fold) and 4 d for PAO1 (5-fold) exposure. No other Pseudomonas strain caused a significant reduction in levels of granulocytes.

pone.0143604.g004: Pulmonary Granulocyte Infiltration during Pseudomonas Exposure.Balb/c mice were endotracheally instilled with saline or 106 cfu of each Pseudomonas strain. At various times following exposure, animals were euthanized and lungs were harvested. Lungs were sectioned and stained with fluorescently tagged Ly6G antibody (inset shows example micrographs). Numbers of positively stained cells (red) were enumerated from nine micrograph fields (three fields from three mice for each treatment) and expressed as fold-change compared to those from saline-exposed mice. Asterisks indicate statistically different values compared to saline exposures, as determined using ANOVA and Dunnett’s Multiple Comparison Test (p < 0.05). Bacteria with significant differences of at least 2-fold are indicated with red boxes in the graph legends.

Mentions:
Certain Pseudomonas strains clearly induced a strong pro-inflammatory response, so pulmonary inflammation was monitored following exposure. This was done by enumerating granulocytes in lung tissue from micrographs such as those shown in insets of Fig 4. All Pa strains, but no other species were able to elevate numbers of pulmonary granulocytes between two and 48 h, and levels stabilized to control values by one week (Fig 4). The response appeared to be separated into two phases; an initial activation that occurred between two and 24 h (PAO1, Pa10752, Pa700370), and a delayed response at 48 h post-exposure (Pa31480, Pa700370, Pa700371). Furthermore, there was statistically significant drop in granulocyte levels at two hours for Pa10752 (2-fold) and 4 d for PAO1 (5-fold) exposure. No other Pseudomonas strain caused a significant reduction in levels of granulocytes.

Bottom Line:
However, Pf and Pp strains were the most antibiotic resistant, with ciprofloxacin and colistin being the most effective of those tested.Serum amyloid A was elevated at ≥ 48 h post-exposure by only some Pa strains.No relationship was observed between strains and levels of peripheral leukocytes.

ABSTRACTPseudomonas species are opportunistically pathogenic to humans, yet closely related species are used in biotechnology applications. In order to screen for the pathogenic potential of strains considered for biotechnology applications, several Pseudomonas strains (P.aeruginosa (Pa), P.fluorescens (Pf), P.putida (Pp), P.stutzeri (Ps)) were compared using functional virulence and toxicity assays. Most Pa strains and Ps grew at temperatures between 28°C and 42°C. However, Pf and Pp strains were the most antibiotic resistant, with ciprofloxacin and colistin being the most effective of those tested. No strain was haemolytic on sheep blood agar. Almost all Pa, but not other test strains, produced a pyocyanin-like chromophore, and caused cytotoxicity towards cultured human HT29 cells. Murine endotracheal exposures indicated that the laboratory reference strain, PAO1, was most persistent in the lungs. Only Pa strains induced pro-inflammatory and inflammatory responses, as measured by elevated cytokines and pulmonary Gr-1 -positive cells. Serum amyloid A was elevated at ≥ 48 h post-exposure by only some Pa strains. No relationship was observed between strains and levels of peripheral leukocytes. The species designation or isolation source may not accurately reflect pathogenic potential, since the clinical strain Pa10752 was relatively nonvirulent, but the industrial strain Pa31480 showed comparable virulence to PAO1. Functional assays involving microbial growth, cytotoxicity and murine immunological responses may be most useful for identifying problematic Pseudomonas strains being considered for biotechnology applications.